subcellular localization of d2 dopamine receptor isoforms · er marker; salamero et al., 1996) was...

INTRODUCTION

The D2 receptor subtype mediates many important effects ofdopamine in the central and peripheral nervous system ofmammals (Civelli et al., 1993; Missale et al., 1998). Thisreceptor is mainly distributed in the nigral and striatal regionof the basal ganglia (where it contributes to sensory and motorcontrol), in the olfactory bulb and retina (where it modulatessensory perception) and in the prolactine cells of the anteriorpituitary gland (where it inhibits prolactin secretion). Like allthe known vertebrate dopamine receptors, the D2 receptorsubtype belongs to the superfamily of G-protein-coupledreceptors, structurally characterized by the presence of seven-transmembrane segments (Gether and Kobilka, 1998; Missaleet al., 1998; Valdenaire and Vernier, 1997). The basicmolecular function of the agonist-bound form of the receptoris to act as an exchange factor for the α-subunit ofheterotrimeric G proteins, the presence of the βγsubunits beingstrictly required (Cherfils and Chardin, 1999; Neer and Smith,1996; Valdenaire and Vernier, 1997). The mammalian D2receptor subtype belongs to the D2 class of dopamine receptors,this latter being functionally, structurally and evolutionarily

very distant from the other dopamine receptor class, D1. As faras we know, the D2 receptor is coupled only to the class ofGαo/Gαi proteins sensitive to pertussis toxin. It elicits asecondary interaction with different effector proteins,especially the Ca2+ and K+ voltage-sensitive channels andadenylyl cyclase (Lledo et al., 1994; Missale et al., 1998).

The mammalian D2 receptor exists in two isoforms generatedby the differential splicing of the pre-mRNA, which modifiesby 29 amino acids the size of the third cytoplasmic loop of thereceptor (Dal Toso et al., 1989; Giros et al., 1989). Thiscytoplasmic loop has been shown to be involved in the couplingof the receptor with heterotrimeric G proteins for several kindsof G-protein-coupled receptors. Thus the two D2 receptorisoforms (D2a, long isoform, and D2b, short isoform) werethought to differentially interact with heterotrimeric G proteinand intracellular signaling pathways (Dal Toso et al., 1989;Giros et al., 1989; Montmayeur et al., 1993). However, despitethe suggestion of a preferential interaction of the D2a isoformwith Gαi2 protein (Guiramand et al., 1995), no obviousevidence of functional differences between the two D2 receptorisoforms have been shown (Huff et al., 1998; Missale et al.,1998; Sokoloff and Schwartz, 1995). The only clear difference

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The dopamine D2 receptor exists as a long (D2a) and a short(D2b) isoform generated by alternative splicing of thecorresponding transcript, which modifies the length of thethird cytoplasmic loop implicated in heterotrimeric G-protein-coupling. Anatomical data suggested that thissegment regulates the intracellular traffic and localizationof the receptor. To directly address this question weused a combination of tagging procedures andimmunocytochemical techniques to detect each of the twoD2 receptor isoforms. Surprisingly, most of the newlysynthesized receptors accumulate in large intracellularcompartments, the plasma membrane being only weaklylabeled, without significant difference between the tworeceptor isoforms. Double labeling experiments showedthat this localization corresponded neither to endosomalcompartments nor to the Golgi apparatus. The D2 receptor

is mostly retained in the endoplasmic reticulum (ER), thelong isoform more efficiently than the short one. It isaccompanied by a striking vacuolization of the ER, roughlyproportional to the expression levels of the two receptorisoforms. This phenomenon is partly overcome bytreatment with pertussis toxin. In addition, an intrinsicactivity of the D2 receptor isoforms is revealed by [35S]-GTPγS binding and cAMP assay, which suggested thatexpression of weakly but constitutively active D2 receptorspromotes activation of heterotrimeric G protein inside thesecretory pathway. This mechanism may participate in theregulation of the cellular traffic of the D2 receptorsisoforms.

Key words: Heterotrimeric G proteins, Cell compartments, Intrinsicactivity

SUMMARY

Intracellular retention of the two isoforms of the D 2dopamine receptor promotes endoplasmic reticulumdisruptionDelphine Prou 1-3,*, Wen-Jie Gu 1,*, Stéphane Le Crom 1, Jean-Didier Vincent 1, Jean Salamero 2 andPhilippe Vernier 1

1DEPSN, UPR 2197, Institut de Neurobiologie Alfred Fessard, CNRS, Avenue de la Terrasse, F91198 Gif-sur-Yvette Cedex, France2UMR 144 CNRS Institut Curie, Laboratoire C Burg, 12 Rue Lhomond, 75005 Paris, France 3Genaxis Biotechnology, Nîmes, France*These authors contributed equally to this work‡Author for correspondence (e-mail: [email protected])

Accepted 26 June 2001Journal of Cell Science 114, 3517-3527 (2001) © The Company of Biologists Ltd

RESEARCH ARTICLE

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between the two isoforms of the D2 receptor is that the amountof each of the corresponding mRNA varies among brain areas(Giros et al., 1989; Guivarc’h et al., 1995; Montmayeur et al.,1991). In addition, the relative abundance of the D2 receptorisoforms is regulated by sex steroid hormones in anteriorpituitary cells as well as in some brain areas (Guivarc’h et al.,1995; Guivarc’h et al., 1998), possibly modifying dopamineresponses according to the physiological states of the organism.These observations suggested that the distribution of the two D2receptor isoforms may be modulated in a tissue-specific fashion.

One of the most important aspects of D2 receptor functionthat may result from the splicing mechanism is a differentsubcellular localization of the protein. Indeed, the differentialdistribution of D2a and D2b mRNAs in the rat central nervoussystem suggested that the sequence of the third cytoplasmicloop could be involved in the targeting of the receptor to thenerve terminals, soma or dendrites. This parameter has not yetbeen taken into account when the question of the differentialactivity of the D2 receptor isoforms has been examined. Moregenerally, little attention has been given to the subcellularlocalization of the receptor proteins inside cells, especiallyafter the transient transfections commonly used to studypharmacological and functional characteristics of receptors. Asa first step to directly address this question we modified byepitope-tagging the sequences of the two isoforms of the ratD2 dopamine receptor and used them for transient transfectionin several cell lines.

MATERIALS AND METHODS

MaterialsRestriction endonucleases, Taq polymerase and plasmid preparationkits were purchased from Promega. The TA cloning kit and pcDNA3vector were obtained from Invitrogen and the DNA sequencing kitfrom USB Amersham. All other chemical reagents were purchasedfrom Prolabo (France) and cell culture reagents were obtained fromLife Technologies unless indicated.

Construction of epitope-tagged D 2a and D2b receptorexpression vectorsSequences corresponding to the c-myc epitope (amino acidsEQKLISEEDL, recognized by the 9E10 monoclonal antibody; Evanet al., 1985) and the VSV-G epitope (amino acids YTDIEMNRLGK,recognized by the P5D4 monoclonal antibody; Kreis, 1986) wereadded downstream from the translation initiation codon of the D2b andD2a rat sequences, respectively, and inserted in the pcDNA3 vector.The chimerical receptor sequences were obtained by PCRamplification with an upstream oligonucleotide encompassing theepitope sequences and the ten first nucleotides of the rat D2 receptorsequence.

To fuse the C-terminal end of each isoform of the D2 receptor tothe GFP protein in the pEGFP-N1 expression vector (Clontech), thetranslation initiation codon of the GFP was mutated to a valine, andthe stop codon of each of the D2 receptor isoforms was mutated to aglycine. Since the cysteine residue that anchored the C-terminus ofthe D2 receptor is the last of the sequence, a spacer sequence made ofseven amino acids (GVCICCI for the short isoform and GVCCGCGfor the long isoform) has been added to avoid, as much as possible, asteric hindrance between the receptor and the GFP. All the modifiedconstructs were checked by full-length sequencing.

Cell culture, transfection and treatmentsThe different cell lines used in this study (COS-7, HeLa, HEK-293

and NG108.15) were maintained in Dulbecco’s modified Eagle’smedium supplemented with 10% (v/v) fetal calf serum and 2 mM L-glutamine and incubated at 37°C in a 5% CO2, 95% air atmosphere.Cells were generally seeded at 4×106 cells/100 mm2 dishes and 6×106

cells/150 mm2 dishes. After overnight incubation, the cells weretransfected with 10 µg DNA, most often by electroporation (Herr etal., 1994) or by the DEAE-dextran/chloroquine transfection protocol(Pari and Keown, 1997).

In the case of HeLa cells and COS-7 cells, co-expression of D2receptors and the Ii, invariant chain of MHC class II (used as anER marker; Salamero et al., 1996) was obtained by transfecting cellswith one of the pcDNA3-D2 recombinant vectors and the pGEM-Iivector, and overexpressed with the T7 polymerase recombinant virustechnique. In some experiments, the D2 receptor isoforms were co-expressed with the D1A dopamine receptor fused to GFP at the C-terminus. The D1A-GFP construct was produced similarly to theD2-GFP constructs.

All cell treatments were performed in the usual culture medium.Brefeldin A (Sigma), which blocks the activity of the ARF proteinand disorganizes the Golgi apparatus (Donaldson et al., 1992) wasused at a concentration of 10 µg/ml for 60 minutes. At different timesafter cell transfection, pertussis toxin (Sigma) was added for 12 hoursat a concentration of 0.1 µg/ml. Tunicamycin (Sigma), a glycosylationinhibitor, was added at 10 ng/ml or 20 ng/ml for 12 hours.Actinomycin D (Sigma), a transcription inhibitor, was used at 10mg/ml.

Functional characterization of the epitope tagged-D 2receptors expressed in COS-7 cellsForty-eight hours after transfection, the cells were washed twice withPBS, harvested by scraping the plates, homogenized by Polytronapparatus in binding buffer (Tris 50 mM pH 7.7 at 22°C, 120 mMNaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2 and 5 mM EDTA), andcentrifuged once at 17,000 g for 20 minutes. The pellets weresuspended in binding buffer, proteins quantified by Bradford protocol(Biorad) and stored at −80°C until use. The specific bindingcharacteristics of tagged and GFP-fused D2 receptors were definedusing [3H]-spiperone as ligand (Amersham). The saturation curveswere carried out with increasing concentrations of [3H]-spiperone and(−)-butaclamol 1 µM to measure the total binding and (+)-butaclamol(RBI) to estimate the nonspecific binding. The binding assays wereinitiated by addition of 50 µg membrane proteins to [3H]-spiperone(0.01-0.90 nM) in a total volume of 1.5 ml binding buffer. Afterincubation for 1 hour 30 minutes at 37°C, the reactions were stoppedby filtration with GF-B glass-fibre filters (Millipore). The filters werewashed twice with ice-cold wash buffer (50 mM Tris pH 7.7) anddried under vacuum. The filters were counted in 10 ml scintillationliquid. Bmax and Kd values were measured after computing the crudebinding values in the Kaleidagraph software (Synergy Software).

For measurements of cAMP levels, transfected cells were seededat 1.25×105 cells in 24 wells cell culture plate. Forty-eight hoursafter transfection, cells were incubated with 0.5 mM of thephosphodiesterase inhibitor, IBMX (3-isobutyl-1-methylxanthine;Sigma), 0.5 mM IBMX and 1×10−5M forskolin (direct activator of theadenylyl cyclase; Sigma) or 0.5 mM IBMX, 1×10−5M forskolin(Sigma) and 1×10−5 M bromocriptine (D2 receptor agonist; Sigma)for 15 minutes at 37°C in 500 µl medium without fetal calf serum.The stimulation was stopped by adding 500 µl of HCl 0.2 N andkept at 4°C until use. The inhibition of forskolin-induced cAMPaccumulation by D2-specific agonist bromocriptine was determinedby competition (Nordstedt and Fredholm, 1990).

Immunocytochemistry and immunofluorescencemicroscopy of the epitope-tagged D 2 receptor isoformsand cellular markersThe transfected cells were grown at low density on coverslips for 48hours before being processed for immunocytochemistry. After three

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3519Subcellular localization of D2 dopamine receptor isoforms

washes in ice-cold PBS, cells were fixed in 3% paraformaldehyde(Sigma) freshly made in phosphate-buffered saline (PBS pH7.4, 0.1mM CaCl2 and 0.1 mM MgCl2) for 15 minutes at room temperature.Cells were incubated in PBS containing 50 mM NH4Cl (Sigma) as ablocking agent for 30 minutes and washed again in PBS. Cellpermeabilization was performed in PBS containing 0.1% saponin(Sigma) and 0.2% BSA (Sigma) for 30 minutes. The permeabilizedcells were then incubated with primary antibodies (1/200) for 60minutes at room temperature or overnight at 4°C in PBS containing0.1% saponin and 3% BSA, and washed three times in PBS. Then,the cells were incubated for 2 hours at room temperature withsecondary antibodies (1/300) and washed three times in PBS. Theanti-D2 receptor antibody (SM) is a rabbit polyclonal antibody raisedagainst the sequence of the third cytoplasmic loop of the receptorfused to GST (Maltais, 2000). The secondary antibodies were eitheran IgG anti-mouse or an IgG anti rabbit immunoglobulin, labeled withTexas-Red or FITC (Cappel) depending on the experiments.

Colocalization experiments were performed to look for precisedistribution of the D2 receptor isoforms with specific antibodies formarkers of intracellular compartments. Polyclonal antibody to Rab6(used as a Golgi marker; Martinez et al., 1994; a kind gift of B. Goud,Institut Curie, Paris), was used in colocalization experiments. Theendoplasmic reticulum was identified by the localization of the p35isoform of the invariant chain of MHC class II molecules, co-transfectedwith the D2 receptor isoforms (Hemar et al., 1995). Early endocytoticcompartments were identified with transferrin directly labeled withrhodamine (a gift from A. Dautry-Varsat, Institut Pasteur, Paris).Internalization kinetics of rhodamine-labeled transferrin were performedat 37°C for 5 minutes, 10 minutes, 15 minutes, 1 hour and 4 hours.

Cells were analyzed with a Leica DMRB microscope using a 40×and 63× fluorescence lenses. Subcellular localization of the markerswas generally performed by double-labeling immunocytochemicalexperiments. Confocal laser scanning microscopy andimmunofluorescence analysis were performed using a TCS4Dconfocal microscope based on a Leica DM microscope interfaced withan Argon/Krypton laser and with an accousto optic tunable filter(AOTF). Simultaneous double fluorescence acquisitions wereperformed using the 488 nm and the 568 nm laser lines to excite FITCor GFP, and Texas Red. The fluorescence was selected withappropriate double fluorescence dichroic mirror and band pass filtersand measured with blue-green sensitive and red side sensitive-onephotomultipliers. The absence of cross detection between theFITC/GFP and Rhodamine/Texas-Red emissions was carefullychecked

Biotinylation of cell membranes for the separation fromthe intracellular compartmentBiotinylation of the plasma membrane was carried out as described(Gottardi et al., 1995). Cells were resuspended in ice-cold DMEM bytrypsinization and washed with Tris-Ca-Mg twice (Tris 50 mM pH7.7 at 22°C, 0.1 mM CaCl2, 1 mM MgCl2 and 5 mM EDTA). Cellswere then incubated with 5 mg/ml NHS-SS-biotin (Pierce) inbiotinylation buffer (10 mM triethanolamine pH 7.5 at 22°C, 2 mMCaCl2 and 150 mM NaCl) twice consecutively for 25 minutes at 4°Cwith gentle agitation. Cells were rinsed twice with Tris-Ca-Mg-glycine (Tris 50 mM pH 7.7 at 22°C, 0.1 mM CaCl2, 1 mM MgCl2,5 mM EDTA and 100 mM glycine) and washed in the same buffer for20 minutes at 4°C with gentle rotation. Cells were then rinsed twicewith Tris-Ca-Mg. Cells were homogenized by 25 passages in aRadnoti homogeneizer and centrifuged at 4000 g for 10 minutes. Thepellet (N: nuclear) was kept and suspended in 500 µl binding bufferfor assays. The supernatants (900 µl in Tris-Ca-Mg buffer) wereincubated with 100 µl of packed streptavidin-agarose beads (Pierce)for 16 hours at 4°C with rotation. The beads were washed twice inTris-Ca-Mg to eliminate unbound membranes (non-biotinylatedmembrane). The eluate was centrifuged at 35,000 g for 1 hour, andthe pellet suspended in 500 µl volume buffer for binding assays. The

membranes bound to the beads (biotinylated membrane) were finallysuspended in 500 µl volume for binding assays. Given the low amountof material recovered by this technique, receptor binding assays of thethree fractions were performed at a single concentration (5×10−8 nM)of [3H]-spiperone.

[35S]-GTPγS binding assayForty-eight hours after transfection, the cells were washed twice withPBS, harvested by scraping the plates, centrifuged at 1700 g for 10minutes at 4°C. The pellet was homogenized by Polytron apparatusin buffer A (20 mM Hepes, 6 mM MgCl2, 1 mM EDTA, 1 mM EGTApH 7,4) and centrifuged twice at 48,000 g for 1 hour at 4°C. Thepellets were resuspended in buffer A, proteins quantified by Bradfordprotocol (Biorad) and stored at −80°C until use.

To perform the [35S]-GTPγS binding (Gardner, 1996) wepreincubated the membrane proteins (30-50 µg) for 30 minutes at30°C with or without drugs (Table 2) ((+)-butaclamol (RBI), (−)-sulpiride (Sigma), bromocryptine (RBI)) in a total volume of 0.9 mlof buffer B (20 mM Hepes, 10 mM MgCl2, 100 mM NaCl, pH 7.4)with 0.1 mM dithiotreitol (DTT) and 10−6 M GDP. The binding assayswere initiated by addition of 100 µl of [35S]-GTPγS (100 pM; ICN).After 30 minutes, the reactions were stopped by addition of 4 ml ofice-cold phosphate buffered saline (0.14 M NaCl, 3 mM KCl, 1.5 mMKH2PO4, 5 mM Na2HPO4, pH 7,4) and by rapid filtration with GF-B glass-fibre filters (Millipore). The filters were washed three timeswith the same buffer and dried under vacuum. The radioactivity of thefilters was determined by liquid scintillation counting.

RESULTS

Functional characterization of modified dopamineD2 receptors transiently expressed in cellsSince the two isoforms of the rat D2 receptor were modified byheterologous sequences added either to the N-terminus or tothe C-terminus, the modified receptors had to be functionallycharacterized. For the N-terminal epitope-tagged sequences,binding assays performed with [3H]-spiperone revealed thathigh levels of D2 receptors were expressed 48 hours aftertransfection in COS-7 cells (300-400 fmol/mg proteins) andthat the ligand Kd of the two receptor isoforms were similarand virtually identical to those of wild-type receptors.Essentially similar results were obtained for the GFP-fused D2receptor isoforms (Table 1).

Accordingly, when stimulated by the agonist bromocriptine(10−5M), these epitope-tagged receptors were as potent as thetwo unmodified receptor isoforms to inhibit foskolin-inducedcAMP accumulation in transfected cells (Table 1). Incidentally,we did not find a significant difference between the twoisoforms for their efficiency to inhibit adenylyl cyclase in COS-7 cells. Therefore, as far as the functional properties of theproteins are concerned, they did not appear to be significantlyaffected by the N- and C-terminal modifications, as reportedfor several others G-protein-coupled receptors and theirepitope-tagged receptors (Barak et al., 1997; Drmota et al.,1998; Liu et al., 1999; Schulein et al., 1998; Tarasova et al.,1997).

Characterization of the detection of the two D 2receptor isoforms after transient expression inseveral cell linesThe subcellular distribution and intracellular transport of thelong (D2a) and short (D2b) isoforms was first analyzed by the

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immunocytochemical detection of the VSV-G epitope and c-myc epitope with specific monoclonal antibodies (seeMaterials and Methods). Forty-eight hours after transfection inCOS-7 cells, the labeling corresponding to the epitope-taggedreceptors was surprisingly found to be localized almostexclusively in intracellular compartments. The plasmamembrane appeared only very weakly decorated and evenundetectable in many cells (Fig. 1A,B). In general, the longisoform seemed to be more strongly accumulated inintracellular compartments than the short isoform. To eliminatethe possibility of an artefact due to receptors overexpression inpeculiar cells, different cell lines were transfected by each of

the two D2 receptor isoforms. In HeLa fibroblast cells (Fig.1C,D) and HEK-293 cells, the pictures obtained werecomparable with those obtained in COS-7 cells. Identical datahave also been obtained in the NG108.15 neuroblastoma-glioma hybrid (data not shown). It suggested that thepredominant intracellular localization depended essentially onthe intrinsic properties of the receptor, and not on the cell typesused for transient expression. The appearance of receptorstaining did not correspond to a delayed transport to the plasmamembrane. Indeed, the overall receptor distribution remainedthe same up to 96 hours after cell transfection, the number oflabeled cells decreasing progressively with time. Treatmentwith the transcription inhibitor, actinomycin D, given 48 hoursafter transfection, did not modify this labeling, indicating thatthe receptors were retained into intracellular compartments.

At this point, several control experiments were undertakento search for some of the problems that may have accountedfor the predominant intracellular detection, somewhatunexpected for a cell surface receptor. Several conditions ofcell fixation, permeabilization and antibody incubation wereused without significantly modifying the labeling appearance.Since the interaction between the N-terminal tags and theglycosyl moieties may have impaired antibody recognition, wetreated the transfected cells by tunicamycin, a glycosylationinhibitor. However, in these conditions, the detection of the D2receptor isoforms at the cell surface was further decreased ina dose dependent manner (Fig. 2) since inhibition of proteinglycosylation prevents the transport of the receptors to theplasma membrane.

Thus we chose to use different anti-receptor antibodies,although these reagents cannot discriminate between the twoD2 receptor isoforms. A first D2-specific receptor polyclonalantibody (a kind gift from B. Ciliax, Emory University) (Yunget al., 1995) gave approximately the same kind of labeling asthat obtained with the anti-tag antibodies (data not shown).By contrast, the pictures obtained with a second polyclonalantibody (see Materials and Methods) directed against the third


Table 1. Functional characteristics of the modified D2 receptors in transfected COS-7 cellsMyc-D2S Myc-D2S-GFP VSVG-D2L VSVG-D2L-GFP

Binding assaysKd (M) 4.10 E-11±0.9 E-11 2.53 E-11±0.5 E-11 3.35 E-11±0.7 E-11 2.50 E-11±0.3 E-11Bmax (fmol/mg of protein) 373.00±101.00 325.00±95.00 329.00±118.00 290.00±127.00

Inhibition of forskolin-induced 45.60±10.5 50.75±9.9 64.40±7.3 66±9.3cAMP accumulation (%)

The Bmax and Kd values for the binding of [3H]-spiperone to membranes of transfected cells are given (see Materials and Methods), as well as the percentage ofinhibition of forskolin-induced cAMP accumulation by the tagged D2 receptors in COS-7 cells. Values are given±s.e.m (n=4). cAMP values were divided by theprotein amount in each well and by the receptor number (Bmax) expressed in COS-7 cells.

Fig. 1. Immunodetection of the epitope-tagged D2 receptor isoformsin transfected COS-7 and HeLa cell lines. COS-7 cells (A,B) andHeLa cells (C,D), have been transfected by either D2b, revealed bythe monoclonal 9E10 anti-myc antibody coupled to Texas-Red(A,C), or D2a, revealed by the monoclonal P5D4 anti VSV-Gantibody coupled to Texas-Red (B,D). These confocal microscopeimages reveal the large predominance of intracellular labelingdetected with these antibodies. Bar, 2.5 µm.

Fig. 2.Effect of tunicamycin on the labeling and thetransport of the D2 receptors to the plasmamembrane. COS-7 cells transfected by the myc-tagged D2b receptor were treated with tunicamycinfor 12 hours at 10 ng/ml (B) and 20 ng/ml (C), andcompared with control cells (A). The receptors werevisualized with the monoclonal 9E10 antibody,coupled to Texas-Red. Note the massive restrictionof the receptor localization close to the nuclearmembrane with increasing doses of tunicamycincompared with control cells. Bar, 3 µm.


cytoplasmic loop of the receptor showed, for the two isoforms,a clear staining at the plasma membrane, although most of theimmunoreactivity was observed scattered inside the cells (Fig.3C).

To verify that the polyclonal anti-D2 antibody (SM)recognized the D2 receptors without any bias (Grayson et al.,1998), we used D2b receptor-GFP fusion proteins (Fig. 3). Thelabeling obtained with the polyclonal anti-receptor antibody(see Materials and Methods; Fig. 3C) completely overlappedwith that of D2b receptor-GFP fusion proteins (Fig. 3A) intransfected cells, whereas monoclonal anti-N terminal tagantibodies revealed preferentially D2 receptors localized inintracellular compartments (Fig. 3B,D). However, the situationwas different for the two D2 receptor isoforms. If thelocalization of the GFP-fused short isoform strikinglyresembled that obtained without C-terminal fusion, the GFP-fused long isoform displayed a weird intracellular fluorescenceconcentrated inside the nucleus. The D2a-GFP and D2b-GFPreceptor mRNA levels measured by northern blots showed thatthe amounts of each of the transcripts were about the samethroughout the time course of transient expression in COS-7cells. Thus it is probable that some degree of degradation ofthe GFP-tagged D2a receptor occurred (data not shown).

To test the possibility of an obligatory interaction betweenthe two D2 isoforms we co-expressed the D2b-GFP constructwith the unmodified D2areceptor in COS-7 cells. Nevertheless,no apparent changes in D2 receptor localization could be seenin these experiments (data not shown).

Identification of the intracellular compartmentswhere the D 2 receptor isoforms accumulateConfocal examinations of the transfected cells confirmed thatmost of the receptor labeling was concentrated in intracellularcompartments; the staining spread over endoplasmic reticulum(ER) and Golgi apparatus. When the N-tagged receptors werelabeled with the monoclonal antibodies, it appeared that thelong D2a isoform is distributed widely over the cell cytoplasm,with a fine-grained aspect, whereas, the D2b isoform isgenerally more densely packed close to cell nucleus (Fig. 1).No significant differences between the two isoforms could be

seen for the localization at the plasma membrane. In addition,given the variability of labeling obtained from one transfectedcell to another, it was difficult to firmly substantiate adifferential subcellular localization for the two receptorisoforms. We used plasma membrane biotinylation of the cellstransfected by each of the isoforms to better quantify theproportion of receptors localized at the plasma membrane andinside the cells. By this technique, 20 to 30% of the receptorsthat bound [3H]-spiperone were retained on the biotinylatedmembranes, with no significant difference between the twoisoforms (Fig. 4). These results confirmed that a large majorityof the two D2 receptor isoforms were held into intracellularcompartments.

To ascertain the precise localization of the two D2 receptorisoforms, the simultaneous visualization of different organellemarkers was examined using double-immunofluorescenceanalyzed by confocal microscopy. These observations werecarried out in COS-7 and HeLa cells. We examined thepossibility that the intracellular localization of the D2 receptorscould correspond to an accumulation in the endocytoticpathway promoted by a large retrieval of the receptors from theplasma membrane. Colocalization of the receptor withrhodamine-labeled transferrin (well known to be endocytosedin clathrin-coated vesicles) was checked at different periods oftime (5 minutes, 10 minutes, 15 minutes, 1 hour and 4 hours)after a 10 minute exposure to this ligand. Either after a shortdelay (5 minutes), when transferrin is essentially localized inperipheral early endosomes, or at longer chase time (60minutes), when transferrin is found concentrated in thepericentriolar early endosomes (Fig. 5C,F), the labeling of theD2 receptors (Fig. 5A,D) and the endosomal marker werealways mutually exclusive (Fig. 5B,E). This observationstrongly suggests that, at least when cells are not exposed toD2 receptor agonist, the receptor is not significantly present inthe clathrin-dependent endocytosis-recycling pathway.

Another possibility was that D2 receptors accumulate in thesecretory pathway, especially in the Golgi apparatus. The Rab6immunoreactivity, used as a marker of the Golgi complex,colocalized poorly with the two D2 receptor isoforms (Fig. 6A-D). It indicated that the receptors were not accumulating

Fig. 3.Comparison of thedistribution of the D2breceptor revealed by antiepitope-tag antibody andGFP-tagged fusion intransiently transfected COS-7cells. The labeling exhibitedby the D2b receptor fused tothe GFP is seen bothintracellularly and at theplasma membrane (A,B) andis very similar to thatobtained with anti-D2receptor polyclonal antibody,coupled to Texas-Red (C). Insharp contrast, the D2breceptor-GFP construct (B)provides a visualizationsignificantly different fromthat obtained with the monoclonal 9E10 antibody, coupled to Texas-Red (D), which revealed mainly intracellular compartments. Confocalmicroscope images; bars, 1 µm.

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significantly in the Golgi sub-compartments characterized bythis low molecular weight G protein. Further evidence for thelocalization of the receptors outside Golgi membranes wasprovided by cell treatment with brefeldin A (BFA). Thisantibiotic disrupts the organization of Golgi saccules andpromotes a relocalization of Golgi proteins in upstreammembrane compartments (Donaldson et al., 1992). In BFA-treated cells, the expected redistribution of the Rab6

immunofluorescence all over the cytoplasm was observed (Fig.6H). By contrast, the intracellular distribution of the twoisoforms of the dopamine receptor was not significantlymodified by the BFA treatment (Fig. 6G).

The localization of D2 receptors in the ER compartmentswas examined with reference to the invariant chain (Ii) of MHCclass II molecules, transfected together with the D2 receptorisoforms (see Materials and Methods). Here, a significantproportion of the receptor labeling appeared to be colocalizedwith the Ii marker (Fig. 7A-D). However, a general observationwas that Ii labeling looked excluded from the sites where thereceptor was the most abundant. The long D2a isoform was, ingeneral, more widely distributed together with the ER markerthan the D2b isoform (Fig. 7A,C). This evidence supported ourprevious observations of the long isoform being retainedfurther upstream and more strongly than the short isoform inearly compartments of the secretory pathway. In addition, asclearly visualized by the Ii labeling, it became obvious that thisretention of the D2 receptors in the ER was accompanied by adramatic change in the ER morphology. This latter appearedgenerally enlarged and transformed in large vacuoles (Fig.7B,D). This morphological change is only seen in D2 receptor-transfected cells and it looked broadly proportional to theexpression levels of the receptors in the cell.


Fig. 4.Relative quantification of the proportion of the D2 receptorisoforms located at the plasma membrane. After membranebiotinylation (see Materials and Methods), the COS-7 cellstransfected by either D2a or D2b receptors were lysed and the wholecell lysate was fractionated by centrifugation to provide a nuclearpellet (N; nuclei and cell debris) and a post-nuclear supernatant. Thissupernatant was submitted to streptavidine chromatography toseparate biotinylated membranes from nonbiotinylated membranes.In the three fractions, the amount of D2a (grey bars) and D2b (emptybars) receptors were quantified by [3H]-spiperone binding (A) andcompared with the total activity of alkaline phosphatase, a marker ofplasma and nuclear membrane (B). The histograms correspond tovalues ± s.e.m (n=3).

Fig. 5.The D2 receptor isoforms are notfound in the transferrin endocytosis-pathway. The intracellular labelingobtained with rhodamine-labeledtransferrin either after 10 minutes (C) or60 minutes (F) incubation at 37°C doesnot match that of the tagged-D2b receptorrevealed by the monoclonal 9E10antibody coupled to FITC (A,D) at thesame incubation time. Superimposition ofconfocal images of the transferrin and D2receptor labeling (B,E) clearly shows thatthe two types of labeling are mutuallyexclusive. Confocal microscope images;bar, 2 µm.


The D2 receptor-induced vacuolization of theendoplasmic reticulum is partly due toheterotrimeric G protein activationThe observation of the large accumulation of the D2 receptorisoforms in intracellular compartments and of the ERvacuolization prompted us to further study the mechanisms ofthis striking effect. The first evidence of a disturbance ofmembrane protein transport was provided by the observationthat co-expression of each of the D2 receptor isoforms togetherwith the D1A receptor almost completely blocked the transportof this latter protein (Fig. 8B), which otherwise is able to reachthe plasma membrane (Fig. 8A). This phenomenon accountedfor a rather general blockage of the intracellular transport oftransmembrane proteins, at least of the pathway used by G-protein-coupled receptors

Since the mammalian D2 receptor is known to be coupled tothe Gαo and Gαi class of heterotrimeric G proteins and sinceactivation of Gαo/Gαi proteins was shown to affect intracellulartransport (Leyte et al., 1992), we decided to examine to what

extent heterotrimeric G protein activation by the expressed D2receptors may account for the modification of the ERmorphology. To do that, we treated the cells with pertussistoxin (PTX), either just after transfection by the D2 receptorisoforms (Fig. 7E,F) or 2 hours after transfection (Fig. 7G,H).Interestingly, only when PTX was applied early after thetransfection, the vacuolization of the ER appeared significantlyovercome. In this condition, it became difficult to see ERmodification, except in the cells that exhibited the highestlevels of receptor expression. Thus, it seemed probable thatsome degree of constitutive activity of the D2 receptor (Hall,1997) was able to turn on the PTX-sensitive G protein presentin the ER or the cis-Golgi. This activation seemed responsible,at least in part, of the ER vacuolization.

In order to test the hypothesis of D2 receptor intrinsicactivity, we performed two kinds of experiments. The firstlooked at the binding of [35S]-GTPγS to indicate G proteinactivation and the second assayed cAMP levels, which reflectthe effect of receptors on signaling pathways. We used a well-

Fig. 6.The D2 receptor isoforms arepoorly co-localized with Rab6, a markerof the Golgi complex, in COS-7 and HeLacells. As analyzed with a confocalmicroscope, the labeling for the D2aisoform obtained with the monoclonalP5D4 antibody, coupled to FITC, in HeLacells (A) and with the monoclonal 9E10antibody, coupled to FITC, for the D2bisoform (C), overlap only for a very smallpart with that obtained with an anti-rab6polyclonal antibody, coupled to Texas-Red, (B,D). The D2a receptor transfected in COS-7 cells displays essentially the same pictures (E,F). This receptor labeling is not significantlymodified by cell treatment with BFA (10 µg/ml) for 1 hour (G), whereas that of Rab6 spreads over the cytoplasm (H). Bar, 2.5 µm.

Fig. 7. The intracellular D2 receptorisoform induces a vacuolization of theendoplasmic reticulum, which isovercome by PTX treatment. In HeLacells, the co-transfection of the Ii invariantchain-coding vector (the Ii chain is usedas an ER marker revealed with a specificpolyclonal antibody; 1/2000) coupled toTexas-Red; (B,D) with either the D2a(A,B) or the D2b (C,D) receptor-encodingvectors, labeled with the monoclonalP5D4 (A) and 9E10 (C) antibodies,respectively and revealed by FITC, showsa partial colocalization of the receptorwith the ER marker. In addition, thepresence of the D2 receptors in theseintracellular membranes promotes adramatic vacuolization of the ER (B,D).When the cells co-transfected by the Iiinvariant chain and the D2a (E,F,G,H) are treated by PTX (0.1 µg/ml; E,F) at the same time as the transfection procedure, only minor alterationsin the ER morphology are seen 12 hours later (F). Conversely, the ER disruption is still obvious (H) when the incubation with PTX begins only2 hours after cell transfection (G-H). Confocal microscope images; bars, 2.5 µm.

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described D2 receptor agonist (bromocriptine) and twoantagonists (butaclamol(+) and sulpiride(−)), which have beenshown to behave as inverse agonist (Hall, 1997). As expected,bromocriptine elicited a significant increase of [35S]-GTPγSbinding (Table 2). By contrast, butaclamol had no effect on thisphenomenon, behaving as a true antagonist. Interestingly,sulpiride clearly decreased [35S]-GTPγS binding at highconcentrations (10−5 M) compared with low concentrations(10−10 M), thus acting as an inverse agonist, demonstrating theintrinsic activity of the two D2 receptor isoforms. Surprisingly,only about half of the value obtained in untransfected COS-7cells (37.3±6.5 d.p.m./µg of protein) was observed in COS-7cells expressing D2 receptor isoforms (14.4±1.1 d.p.m./µg ofprotein) for the D2b and 22.4±1.8 d.p.m./µg of protein for theD2a). When cAMP levels were assayed in COS-7 cells (Table2), we consistently observed that the level of cAMP in COS-7cells expressing the D2 receptors was about half of that inuntransfected cells (32.7% for the D2b and 46.1% for the D2a).Nevertheless, activation of D2 receptors by bromocriptinedecreased forskolin-stimulated cAMP accumulation (16% forthe D2b and 32% for the D2a).

DISCUSSION

This work was intended to analyze in detail the subcellularlocalization of the mammalian dopamine D2 receptor in severaltypes of heterologous cells. Our initial aim was to detectdifferential distribution in cell compartments and differentialregulation between the two splicing isoforms of the receptor.Indeed, besides their basic function ofmodulating the activity of heterotrimeric Gproteins, the precise subcellular localization ofmembrane receptors are likely to be majordeterminants of their physiological role(Valdenaire and Vernier, 1997). Thedemonstration that the differential splicing ofthe D2 dopamine receptor is regulated inmammals (Guivarc’h et al., 1995; Guivarc’h etal., 1998) and the existence of a differentiallocalization of the two transcripts in thebrain suggested possible differences in theintracellular targeting of this physiologicallyimportant receptor.

The observation of a predominantly intracellular distributionof the transfected D2 receptor isoforms was very surprising atfirst glance and raised many embarrassing questions. Theproblem is not the strong accumulation of the D2 receptors inthe secretory pathway, but mainly the default of membranelocalization. Some technical issues certainly accounted for thepoor detection of the N-terminal tags when the receptors arelocalized at the plasma membrane. It does not depend on theantibody itself since this phenomenon occurred either with the9E10 anti-myc monoclonal antibody (labeling the shortisoform of the D2 receptor) or with the P5D4 anti-VSV-Gmonoclonal antibody (used to detect the long D2 receptorisoform). The reasons for this are not clear, although somekind of interaction between the receptor N-terminus andcomponents of the extracellular matrix may prevent antibodiesfrom good access to the epitopes. The use of differentapproachs to visualize receptors allowed us to partly solve thisissue. The polyclonal anti-D2 receptor antibody, which isdirected against the third cytoplasmic loop of the receptor,provided a much better plasma membrane decoration than themonoclonal antibody. The short isoform of the D2 receptorfused to GFP gave essentially the same labeling pattern as thatof the anti-D2 receptor antibody, suggesting that they bothprovided a faithful picture of the subcellular localization of thereceptor isoforms.

The main feature of the D2 receptor localization remains thatit is predominantly found intracellularly, corresponding toabout 75-80% of the translated receptors (Fig. 4). Transientexpression of the G-protein-coupled receptors certainlyoverloads secretory intracellular compartments. However, it


Fig. 8.Localization of the D1A receptor isoform co-transfected with the D2b receptor inCOS-7 cells. D1A-GFP fusion construct expressed in COS-7 cells is mainly present atthe plasma membrane (A; confocal microscope image). In cells where it is co-expressedwith the D2b receptor, it appears retained into altered intracellular compartments (B).The co-expressed myc-tagged D2 receptor is visualized with the monoclonal 9E10antibody coupled to Texas-Red (C). Bars, 2 µm.

Table 2. Constitutive activity of the D2 receptors expressed in COS-7 cellsD2b D2a Control

Bmax (fmol/mg of protein) 1302.00±89.0 1560.00±103.0 –

[35S]-GTPγS binding (%)Bromocriptine stimulation (10−10 M to 10−5 M) 14.6±6 4.6±5 –Butaclamol(+)- inhibition (10−10 M to 10−5 M) 0.5±3.2 0.1±3.2 –Sulpiride(−)- inhibition (10−10 M to 10−5 M) 29.6±9 26.1±3.9 –

cAMP accumulation (pmol/mg of protein)Basal 12.1±4* 9.7±2* 18±6*FSK 82.5±32 99±29 78.5±21 FSK+Bromo 69±22 67±18 70±16

The Bmax and Kd values for the binding of [3H]-spiperone to membranes of transfected cells are given (see Materials and Methods). Percentage of stimulationor inhibition of the [35S]-GTPγS binding in COS-7 cells for different treatments are indicated and the cellular levels of cAMP are expressed in pmol/mg of protein(n=3-8). FSK, forskolin-treated cells; FSK+Bromo, forskolin+bromocriptine-treated cells.

*P< 0.05 versus control.


does not prevent a large proportion of the receptors fromreaching the plasma membrane in the case of the D1 dopaminereceptors (Fig. 8) or β-adrenoreceptors (Von Zastrow et al.,1993) or α1β-receptors (Fonseca et al., 1995; Hirasawa et al.,1997). However, in some instances, a predominant intracellulardistribution has been described for a few receptors of the G-protein-coupled receptor superfamily such as the α2C-adrenoreceptor (Daunt et al., 1997), the α1A-adrenoreceptor(Hirasawa et al., 1997), the 5HT1B receptor (Langlois et al.,1996) and the thrombin receptor (Hein et al., 1994). Whetherthese observations have a common mechanism and whetherthey correspond to a natural situation is not known yet.

In the case of the D2 receptor, several hypothesis may haveaccounted for this puzzling observation and some of them havebeen tested in the present study. First, this phenomenon is notcell-specific since it has been observed in HeLa, COS-7, andHEK-293 cells, as well as in the NG108.15 neuroblastoma-glioma hybrid. Second, it does not depend on a delayedtransport to the plasma membrane, as shown by the labelingnot being altered with time. Incidentally, it is worth mentioningthat the two isoforms showed no significant difference in theirpresence at the cell surface, thus indicating that the alternativesplicing is not affecting the protein targeting to the plasmamembrane. By contrast, the longest of the D2 receptor isoformsis more strongly retained in the early secretory membranes, itsdistribution remaining essentially confined to the ER. Therelative blockage of the D2a isoform in the ER was also seenin CHO cells by Fishburn et al., who showed that the long D2areceptor isoform exhibited a glycosylation pattern reminiscentof poorly transported membrane proteins (Fishburn et al.,1995).

Three observations provided clues to explain theintracellular retention of the D2 receptor isoforms. The first oneis that a glycosylation defect, due to an imperfect folding ofthe protein, could theoretically promote a fast retrieval of thereceptors from the intermediate compartment of the Golgicomplex (Gahmberg and Tolvanen, 1996). In the case of theD2 receptors (Fig. 2), a defect in the maturation of thepolysaccharide moities of the receptor appears to be theconsequence of impaired transport out of the ER, but not itsinitial cause, in agreement with previous studies (Fishburn etal., 1995).

The second possibility is that the retention of the receptor inthe ER may be dependent, at least in part, on the activation ofPTX-sensitive G proteins by constitutively active D2 receptors.The existence of a significant intrinsic activity of the D2receptors was first suggested by Hall and Strange (Hall andStrange, 1997). This hypothesis is supported by data showingthat activation of PTX-sensitive-heterotrimeric G proteins wasable to block the formation of secretory vesicles from the TGN(Leyte et al., 1992). A similar mechanism may have accountedfor the impaired transport of the long isoform of the D2receptor early in the secretory pathway. This contention isfurther supported by the fact that this receptor localization isinsensitive to BFA, suggesting that the immature receptors areretained in a membrane compartment that could be excludedfrom the Golgi bi-directional traffic. In addition, the transportof receptors otherwise normally present at the cell surface(such as the D1 dopamine receptor), is also impaired by thesimultaneous presence of the D2 receptor (Fig. 8). Thisindicates that the modification of the membrane protein traffic

induced by the D2 receptor is more general and that it affectsat least one other polytopic transmembrane protein. Whetherthis phenomenon may be elicited by other G-protein-coupledreceptors retained intracellularly is not known. However, in thecase of GABAB R1 subunit which, alone, is both unable to goto the plasma membrane and unable to activate G proteins, noperturbation of the ER or other membrane compartments areelicited (Couve et al., 1998).

A third possibility suggested by a recent study (Vickery andvon Zastrow, 1999) that provides evidence for a constitutiveendocytosis of the D2 receptor in a dynamin-independent,clathrin-independent pathway, as analyzed by the endocytosisof antibodies directed against N-terminal tagged receptor. Ourdata do not exclude this possibility and two of the observationsmade by these authors fit with our own data: (1) thatconstitutive endocytosis is very likely to correspond to aconstitutive activation of the receptor; and (2) an accumulationof the D2 receptors inside the cells is also observed in theseexperiments. However, in the steady-state conditions we used,most of this intracellular accumulation predominantlycorresponded to a blocked transport in the biosyntheticpathway (as supported by tunicamycin treatment) and not toconstitutive endocytosis.

From a different perspective, the poor localization of the D2receptors at the plasma membrane may rely on the lack of acomponent, a molecular partner that would be required for themaintenance of the receptor at the plasma membrane in thetransfected cells. In particular, heterologous receptordimerization should be a requirement for a proper targeting tothe plasma membrane, as recently shown for the GABAB andGABAC receptor subtypes (Kaupmann et al., 1998; White etal., 1998). Although the possibility of self-dimerization of theD2 receptor has been reported by some authors (Ng et al.,1996), no evidence exists for the association of the D2 receptorwith another type of G-protein-coupled receptor or even for anassociation between the two D2 receptor isoforms (this study).In addition, the requirement of some type of ‘scaffoldingproteins’ may be envisaged, such as PDZ-domain proteins, butno consensus for PDZ binding is found for the D2 receptor.

Although the previous hypothesis may account for theunusual localization of the D2 receptors after transientexpression in cells, the provocative observation of a massivevacuolization of the ER is related, at least in part, to receptor-dependent activation of heterotrimeric G proteins. Thesalutatory effect of PTX, based on the ER morphologyanalysis, implies that G protein activation has been elicited bythe endogenous activity of the D2 receptors. The mechanismof PTX inhibition of G protein stimulation relies on theimpairment of the direct interaction of the receptor with the C-terminus of the α subunit of the G protein which is ADPribosylated by the toxin. The phenomenon strongly resemblesthat promoted by the pore-forming toxin aerolysin (Abramiet al., 1998). Although the mechanisms of vacuolizationpromoted by aerolysin are not completely understood, the toxinaffects early steps of protein secretion, as did the dopamine D2receptors. In addition, it involves pertussis-toxin sensitive Gproteins and certainly calcium release from internal stores(Krause et al., 1998). In this respect, the D2 receptor, whichcan also modulate calcium entry via G protein activation(Lledo et al., 1994), may be similar to this pore forming toxin.

The observation of the intracellular localization of the D2

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receptor isoforms raised the question of its physiologicalrelevance. In this respect, a predominant intracellularlocalization of the D2a isoform has been described in thestriatum (Khan et al., 1998), and can also be seen in the paperby Hersch et al. (Hersch et al., 1995) or Delle Donne et al.(Delle Donne et al., 1997). Therefore the regulation of themRNA splicing of the D2 receptor which has been observedin several physiological situations (Guivarc’h et al., 1995;Guivarc’h et al., 1998) may result in a differential localizationof the isoforms in intracellular compartments. Whether itpromotes solely a modification of the receptor trafficking or alsodifferential interactions with unknown regulatory componentsof receptor activity will now need to be carefully investigated.

What could the effect of an intracellular receptor be? Inaddition to heterotrimeric G proteins, important components ofintracellular signalling pathways such as adenylyl cyclase arepresent in the ER and the Golgi apparatus (Yamamoto et al.,1998). Thus, it is plausible that receptors play a role in theintracellular compartments. In addition, modulation ofadenylyl cyclase is only part of the potential effects of the D2receptors in cells. For example, interaction of D2 receptors withheterotrimeric G proteins in the ER and the Golgi may be aregulation factor of the secretion of cell products (Takizawa etal., 1993). In this respect, the D2 receptor is a well knowninhibitor of the secretion of peptidic hormones such asprolactin or GH in the pituitary (Missale et al., 1998). Whetheractivation of the D2 receptor can block not only depolarizationand calcium-dependant hormone release but also other steps oftransmitter secretion would be an attractive hypothesis to test.

This work was supported by grants from CNRS, University Paris-Sud, Institut Universitaire de France, and Association pour laRecherche sur le Cancer. Many thanks to S. Maltais and B. Ciliax forproviding us with anti-D2 receptor antibodies, S. Père for technicalassistance and B. Allinquant for helpful discussions.

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subcellular localization of d2 dopamine receptor isoforms · er marker; salamero et al., 1996) was...

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